In a Drosophila melanogaster and other insects, oxygen is supplied to the tissues by the tracheal system, a network of branched epithelial tubes that opens to the outside at the spiracles and extends branches throughout the body. Extensive arrays of fine terminal branches reach the tissues and transport air directly to the sites of utilization. Terminal branches are formed in an intriguing way by tracheal cells located at the ends of major tracheal branches: each terminal cell sends out many long, branches cytoplasmic extensions that subsequently "hollow out" forming dozens of fine tubules. Formation of terminal branches is induces by signals from oxygen-starved tissues, and the new branches grow out and cover the tissue. Little is known though about the chemical nature of the signals or how they are transduced into an effect on terminal branch outgrowth. The long term objectives of this work are to identify the tracheogenic signal(s), to determine how signal production is regulated by tissue oxygen need, to understand how the signal is received and transduced by the terminal tracheal cells, and to elucidate the subcellular mechanisms that drive cytoplasmic outgrowth and branch formation. We have identified three genes, pruned/DSRF, trimmed, and misguided, that are specifically required in this process. One of these, pruned/DSRF, is the homologue of mammalian serum, response, transcription factor which functions as part of a growth factor regulated transcription complex. pruned/DSRF is specifically expressed in the terminal cells throughout the period of terminal branching; in pruned/DSRF mutants, the terminal cells are present but they fail to form cytoplasmic extensions and the terminal branches of the tracheal tree are strikingly absent. It is proposed that pruned/DSRF protein is part of a regulated transcription complex in terminal cells that is activated by a tracheogenic signal secreted by oxygen-requiring target tissues. The specific aims of this project are: 1. To analyze the effects of constitutively activated forms of pruned/DSRF expressed in terminal cells, in order to test the hypothesis that pruned/DSRF is part of key regulated step in terminal branching. 2. To determine if pruned/DSRF functions in a Ras/Map kinase signalling cascade as does SRF, by expressing activated and dominant negative forms of Ras/MAPK signalling components in the terminal cells. 3. To clone and molecularly characterize trimmed and misguided, two genes required for terminal branch formation and path finding, and to determine their roles in these processes and their functional relationships to pruned/DSRF. 4. To carry out additional genetic screens to identify other genes that are required for terminal branch formation and outgrowth, to determine the cellular defects in the mutants, and to functionally order the new genes with respect to pruned/DSRF and other genes that are involved in the process.